The destruction of tissue in inflammation caused by non-immunological and immunological processes induces the formation of different endogenic substances (mediators and hormones). They regulate the complex steps of activation of the inflammation and tissue regeneration processes. The mediators are formed either by limited and regulated proteolysis of plasma and serum protein factors as humoral mediators; or they are liberated by active secretion and/or cell lysis from cells and tissues as cellular mediators. The mediators and hormones are especially important as specific carriers of chemical information which are formed and secreted by leukocytes in the course of cell proliferation processes (mitosis processes). They are components of the body's defence system whose systemic and local activation they regulate. The mediators contribute to the removal and detoxification of destroyed body's own components and/or intruded foreign components. In addition, by regulation of cell proliferation and tissue growth processes in wound-healing, they contribute to the restoration of physiological functions of the organism. As the classical hormones of endocrine glands, inflammatory mediators are trace components of tissues or blood and are present in very minute concentrations only. Experimental evidence shows that only up to 5,000 of such mediator protein molecules can be maintained in a steady state equilibrium by a cell in the mitotic cycle in its surrounding medium.
A reaction by which cells and organisms are mobilized and transferred by chemical substances from their production and storage sites to sites of functional readiness is defined as "chemorecruitment".
The mobilization of the different individual types of leukocytes and their precursors by chemical substances from their production and storage sites in the bone marrow and their recruitment into blood circulation is represented by two different reactions in vivo. These two reactions are typical classical reactions of pathology; see D. J. Boggs, Ann. Rev. Physiol. 28 (1966), p. 39 to 56; V. Schilling, ed., "Das Blutbild und seine klinische Verwertung", Gustav Fischer Verlag, Jena, 1929.
These reactions are the reactive increase in the number of circulating leukocytes (leukocytosis reaction) and the reactive change of the cell differential of individual circulating leukocyte types (leftward shift reaction). The latter is a result of the increase in the number of juvenile leukocytes. Several types of circulating leukocytes exist: Mature, fully differentiated granulocytes (segmented neutrophilic, eosinophilic and basophilic phagocytes) and mononuclear leukocytes (monocytic phagocytes and lymphocytes) with their different subpopulations (T, B-cells etc.). Of these circulating cells, only the mononuclear leukocytes are capable of further proliferation and differentiation. Precursors of these mature leukocyte types within the development and maturation line (hematopoesis) of the segmented granulocytes (leukopoesis) are immature cell types, namely the neutrophilic bands as well as adult and juvenile metamyelocytes which develop from the myelocyte, promyelocyte, myeloblast and the undifferentiated bone marrow stem cell. The names of the cells within such a development and maturation line are standardized today according to an international nomenclature; see E. L. Peirson, Amer. J. Med. Technol. 42 (1976) p. 288-296.
Endogenous chemical substances which activate and granulate such processes of cell development and maturation are called leukopoetins; see H. E. Whipple and M. I. Spitzer eds., Leukopoesis in Health and Disease, Ann. N.Y. Acad. Sci., 113 (1964), p. 511 to 1092. Leukopoetins which catalyze the release of the different leukocyte types and their precursors from the production and storage sites of the bone marrow into blood circulation are defined as "chemorecruitins" or "leukorecruitins". Their target is the barrier betweeen bone marrow production and storage sites on the one hand and blood circulation of the other hand.
The recruitment of leukocytes from the bone marrow is a characteristic in common to all inflammatory processes, e.g. in bacterial infections, tumors or myocardial infarction, etc.
The leukocytosis and leftward shift reactions are part of cybernetic loops of the body's defence system, which maintain the structural and functional readiness of the organism for tissue repair. Such reactions have often been used as diagnostic means in pathology. Apart from infarctions, immune reactions and mechanical tissue damage, other factors can lead to a leukocytosis reaction. Such factors are psychic stress and heavy meals.
Thus, chemorecruitment reactions must be divided into two different types: Mobilization of immature and mature leukocytes from production (poietic or primary) storage sites on the one hand; and recruitment from different (marginal or secondary) storage sites in tissues, e.g. the spleen, on the other hand. In the latter, mainly adult, mature cell types are recruited; see Boggs, loc. cit. Such leukocytes in secondary storage sites in tissues can also be activated, e.g. by cortisone; see Boggs, loc. cit. The mobilization of leukocytes from both storage sites can lead to a leucocytosis reaction. Therefore, an indication as to the kind of storage pool which has been activated can be obtained by distinguishing young immature and mature leucocytes; see Boggs, loc. cit.
Two negative feedback mechanisms have been postulated for the maintenance of the normal constant concentration and constant population differential of leukocytes in blood. One mechanism is supposed to be responsible for the production of the cells in the bone marrow, the other one for the release of the cells into the blood; see D. R. Boggs, loc. cit., H. E. Wipple and M. I. Spitzer, loc. cit. In these regulatory loops, humoral mediators are said to play a role; see V. Menkin, Biochemical Mechanisms in Inflammation, 2nd edition, Charles C. Thomas, Springfield, Ill., 1956.
V. Menkin, loc. cit. was the first to show that soluble tissue fractions participate in mechanisms which reactively increase the concentration of leukocytes in the blood above the normal range. He also isolated, from inflammatory exudates, a crystalizable substance whose nature has not been characterized in detail. However, the substance elicited a leukocytosis reaction in vivo.
Contamination with bacterial endotoxins presumably simulated part of the activities ascribed to the preparations; see Boggs, loc. cit. Such foreign substances as endotoxins are very effective in many respects on hematopoesis; see O. Luderitz; Angew. Chemie 82 (1979), p. 708 to 722. Thus, it is known that, on the one hand, endotoxins can activate blood plasma protein systems (for instance the kinine and complement protein systems. On the other hand, they have a mitogenic effect on mononuclear leucocytes (B-cell mitogen); see J. Anderson et al., J. Exp. Med. 137 (1973), p. 943 to 953.
In the past, a major subject of research was the development of reliable in vitro test systems for leukocyte recruitment, the results of which would allow to be correlated to in vivo test systems of leukocytosis and leftward shift reactions. A. S. Gordon et al., Ann. N.Y. Acad. Sci., vol. 113 (1964), p. 766 to 789 developed such a laborious in vitro test system. With this system they obtained a humoral plasma factor inducing leukocytosis. Its nature was not elucidated in detail and its function must rather be considered part of the feedback mechanisms which can correct decreased blood leukocyte concentrations (leukopeniae) to normal. K. Rother, Eur. J. Immunol., vol. 2 (1972) p. 550 to 558 developed another, simpler in vitro test system to prove the mobilization of leukocytes from the bone marrow. In vivo, leukocytosis and leftward shift reactions are measured quantitatively by time-dependent periodic counting and differentiation of the different types of leukocytes in the blood after administration of the solution of the substance to be examined; see Schilling, loc. cit.
As a consequence of these findings, humoral serum proteins were prepared which induce the release of leukocytes from an isolate rat femur in vitro and a leukocytosis reaction in vivo. However, they were neither defined in more detail, nor are they molecularly uniform and/or biologically specific. Such a non-specifically acting humoral serum protein was for instance prepared by B. Gehbrehiwet and J. H. Muller-Eberhardt, J. Immunol., vol. 123 (1979), p. 616 to 621, by cleaving a preparation of the complement component C3 by means of trypsin.
A natural humoral leukocyte-recruiting protein ("serum-leukorecruitin") which is different therefrom and which has biological and topochemical specificity, was prepared from contact-activated serum by J. H. Wissler et al. Z. Physiol. 361 (1980) p. 1358.
All above-described preparations for the induction of the leukocytosis reaction are serum-derived (humoral) chemical substances. Cellular mediators (wound hormones), i.e. mediator substances secreted by cells, which cause a leukocytosis reaction (chemorecruitins) have not yet been described. Furthermore, mediator substances (chemorecruitins)--either of humoral or cellular origin--which induce a leftward shift reaction are not known at all.
It is therefore a primary object of this invention to provide a new class of cellular chemorecruitins from leukocytes.
It is another object of this invention to provide a new class of cellular chemorecruitins from leukocytes in highly purified form.
It is another object of this invention to provide a new class of cellular chemorecruitins from leukocytes in physical quantities for practical use.
It is another object of this invention to provide a new class of chemorecruitins from leukocytes, which represent biologically specific, active and naturally acting mediators of the leukocytosis and/or leftward shift reactions.
It is another object of this invention to provide a new class of chemorecruitins from leukocytes, which are suitable for specifically influencing the defense state of mammalian (e.g. human) organisms.
It is still another object of this invention to provide a process for producing and obtaining a new class of chemorecruitins from leukocytes in an economical, biotechnically useful and relatively simple manner.
It is still another object of this invention to provide a process for producing and obtaining a new class of chemorecruitins from leukocytes in a highly purified, molecularly homogenous form and in physical quantities for practical use.
It is still another object of this invention to provide a pharmaceutical composition for specifically influencing the defence state of the body of mammalians.
These and other objects and advantages of the present invention will be evident from the following description of the invention.